The present invention relates to the production and stabilization of functional biological materials, such as proteins, viral agents, or other biological agents or products, including, but not limited to, enzymes, hormones, growth factors, structural proteins, tumor suppressor agents, nucleic acids and nucleic acid probes, vaccines, antigens, antibiotics, lipids, simple and complex carbohydrates, alcohols and other solvents and the products of those methods. Such functional biological materials are useful for the manufacture of vaccines and diagnostic assays or tests, for example.
The production of viruses, viral antigens, and other viral products useful for manufacturing of products such as vaccines and diagnostic assays, however, is expensive, time consuming and requires a high level of technical expertise. Viruses must be propagated in living eucaryotic cells. Eucaryotic cells that can be infected with a particular virus are said to be xe2x80x9cpermissivexe2x80x9d for that virus. However, most eucaryotic cells are not permissive for any given virus, and no techniques exist to predict permissiveness. Additionally, cells that are permissive for virus production may have different levels of permissiveness. Some cell lines may produce large amounts of virus while others may permit only a low level of viral replication. Therefore, to determine the optimal cells for virus production, a sometimes large and often time-consuming empirical study of many cell lines must be performed. Further complicating matters, the optimal cell line may not be from the host (e.g., human cells for viruses affecting humans) from which the virus was obtained. Frequently, no suitable cell line exists for a virus from a particular host.
Cell lines are preferable to primary cell cultures from a host because of their stability, immortality, known characteristics, and behavior that has been defined by long experience. The requirements for use of such cell lines in viral production has led to development of a large number of exotic cell lines that are used to produce various viruses. For example, Canine Distemper Virus (CDV), a morbillivirus, grows well in Vero cells (i.e. African Green Monkey kidney cells) but is not propagated in cells from dogs, while Bovine Leukemia Virus (BLV) is produced from a cell line derived from bat lung tissue.
Unfortunately, growth of viruses in cell lines far removed from the host and tissue normally infected may cause such viruses to act in a fashion that is far removed from their normal behavior.
Further, the complex pathology for the virus can make the manufacture of viruses extremely difficult. For example, CDV and Human Measles Virus (HMV) are closely related viruses. Both viruses act similarly when infecting their respective canine and human hosts. Both produce encephalitis and long-term sequelae in which the central nervous system is damaged or destroyed. The central nervous system disease in dogs that occurs many years after initial CDV infection is called old dog encephalitis (ODE). In humans, the corresponding condition is subacute sclerosing panencephalitis (SSPE). The cause of ODE and SSPE is directly related to the number of virions produced and cells affected during the encephalitic phase of the disease. This in turn is directly related to permissiveness of the brain cells and the ability of the host""s immune system to rapidly respond to the virus. Dogs or humans whose brain viral titers reach exceptionally high levels may develop these long-term consequences many years later. In fact, the high viral titers in some infected patients can result in permanent production of a viral protein in brain cells, even after the infection has cleared. The immune system recognizes the viral protein as foreign and continues to attack it even though no viable virus has been produced for many years. Eventually the combat between the host""s immune system and these aberrant cells creates enough damage to destroy the host""s cognitive capacity. Dogs with ODE are usually euthanized. The outcome of SSPE in humans is a progressive dementia eventually followed by death. Because of this complex disease pathophysiology, use of manufactured viruses and viral products has been unsuccessful in stimulating immunity in vivo.
Since many viruses (such as CDV and HMV) infect brain cells, the permissiveness of brain cell lines is important in studying the pathophysiology of the resultant disease and in producing viruses for diagnostics and vaccines that exactly mimic virus characteristics during natural infection. However, few neural cell lines exist, and they only produce low levels of CDV or HMV. Therefore, these cell lines produce inadequate levels of the virus for study and are especially unsuitable for antigen or vaccine production. A method of modifying such cells or cell lines, and the cell lines themselves, is thus required that will support replication of viruses and viral products to high levels.
In addition to the problems associated with producing known viruses to useful titers, it is extremely difficult to search for an unknown virus because the cell type required for replication of the unknown virus is itself unknown. For example, there are a number of diseases of the central nervous system that may well be caused by undiscovered viral agents. The degenerative diseases of Creutzfield-Jacob Disease (CJD, affecting humans), scrapie (affecting sheep), and bovine spongiform encephalopathy (affecting cattle) all are very slow neurodegenerative diseases whose etiology remains unknown. To date, no causative agent for these diseases has been identified nor has any viral agent been propagated from infected human or animal brain tissue. This has lead to the promotion of various unorthodox hypotheses concerning disease etiology, including suggestions that a special protein (called a xe2x80x9cprionxe2x80x9d, suggested to be an agent capable of causing infection and reproducing without any genetic material) might serve as the infectious agent. However, no cell line that produces prions, even to high levels, has been shown to be infectious. Transgenic mice modified to produce prions are not infectious even when they present the diagnostic hallmarks of the disease. Thus, without means for identification of the infectious agent, no good diagnostic test can be produced for these diseases, and the production of a vaccine is impossible.
Recently, the need for improved ways for development of diagnostic tests and potential vaccines has become of pressing importance. For example, recently cattle in Great Britain were fed meal consisting of sheep and other animal offal. Bovine spongiform encephalopathy was subsequently recognized for the first time in these cattle. A number of humans were infected by eating meat from the infected cattlexe2x80x94constituting a route of infection which had not been previously recognized (now called xe2x80x9cnew variant CJDxe2x80x9d, or nvCJD). Besides the tragic consequences to the infected humans, the slaughter of cattle caused massive economic damage. The finding of nvCJD also produced political repercussions involving the import, export and sale of food and other animal products that might come from infected cattle. Accordingly, the development of a good diagnostic test is required along with the production of an efficacious vaccine. To accomplish these and similar goals, permissive cell lines are required that will allow the propagation of neutrotrophic agents responsible for the etiology of slow dementias like CJD and nvCJD.
As the average age of a nation""s population increases, the incidence of disease states like Alzheimer""s also increases. Alzheimer""s disease is a slowly progressing dementia necessitating difficult, long-term care for the patient. Costs associated with such long-term debilitating diseases can be devastating. Alzheimer""s is not thought to be infectious. However, certain features of Alzheimer""s have caused speculation that infection with an unconventional viral agent (similar to nvCJD) might cause the disease. It may also be possible that a neurotrophic agent, like HMV or related morbillivirus-like viruses, cause the slow disease process that destroys mentation. It Alzheimer""s were caused by an infectious agents, this would have huge consequences for the management, prevention, diagnosis and cure (if possible) of the disease. Then, for example. prevention of Alzheimer""s would likely require the development of a good vaccine. However, as is the case with other dementias produced by unconventional agents (such as ODE and SSPE), no definitive diagnostic test exists. Isolation of a virus from infected brain tissue would thus be a landmark step in diagnosing and possibly treating humans with this disease. Hence, cell lines that are more permissive for viruses like measles may allow the isolation of neurotrophic agents (like CDV and HMV) not yet discovered.
Production of proteins from eucaryotic cell lines using recombinant DNA technologies for vaccines, diagnostic kits or other purposes has the same limitations and problems associated with virus production. Recombinant proteins must be produced by eucaryotic cell lines to assure proper folding, glycosylation, and other constitutive factors that are critical to proper function and stability of the protein or its immunogenicity as a diagnostic reagent or vaccine. The ability to produce these proteins in large quantity while maintaining the correct conformation is difficult. In many cases, a recombinant protein may be produced in cell lines at acceptable levels, but due to some often subtle change in conformation, it is either non-immunoreactive as a diagnostic antigen or vaccine or fails to perform its desired function. For example, Canine Parvo Virus (CPV) was genetically cloned and produced in a eucaryotic cell line at levels which would be commercially feasible. Dogs vaccinated with these recombinant antigens responded by producing antibodies that could, in vitro, neutralize infectious (wild type) parvovirus. Unfortunately, all dogs vaccinated with the recombinant antigen died upon exposure to infectious CPV. It is likely that some slight conformation change prevented the recombinant vaccine from protecting the dogs.
Hence, the production of recombinant proteins is expensive, technically demanding, time consuming and very inefficient. In many cases, recombinant proteins may have useful effects but cannot be used because of the cost involved in their production. For example, numerous anti-tumor peptides are known to have beneficial effects, including apparent reduction or curing of cancers (for example, angiogenesis blockers). It has been recently claimed that angiostatin and endostatin can cure cancers in mice. However, the cost of producing these peptides (which may run as high as $5-20 million for the quantity of agent necessary for a single treatment regimen) prevents their use in cancer therapy. Thus, a cost-effective method of producing recombinant peptides or proteins that maintains their function (enzymic, antigenic, or other functional properties) is desperately needed.
Many constitutively produced proteins and peptides have closely associated xe2x80x9chelper proteinsxe2x80x9d which help induce or maintain proper shape. These helper proteins are often call xe2x80x9cchaperonesxe2x80x9d because they accompany the proteins through the production process. Some of these chaperone proteins bind to other proteins to prevent denaturation (loss of conformation) or other deterioration due to environmental stress. For example, the heat shock proteins (such as hsp70 and hsp90) are produced by cells in response to higher than normal levels of heat. Such heat shock proteins bind to other proteins within a cell, stabilizing them and thereby helping to maintain correct conformation and function of the bound protein. These and other, similar proteins that are produced in response to other stresses are in general called stress proteins.
The structure and functional role of such stress proteins appears to be highly conserved throughout nature, where the various stress proteins appear to play similar chaperone roles in both procaryotic and eucaryotic cells. This has lead to a large number of studies of and proposed uses for such proteins. For example, a number of works in the literature describe uses for such proteins based on in vitro contact of various biological materials with exogenously-produced stress proteins. These works, which are summarized below, are incorporated herein by reference:
Neupert et al. (U.S. Pat. No. 5,302,518) suggest that proper folding of proteins may be mediated in vivo by constitutive heat shock proteins, such as GroEL and hsp60 (which occur in E. coli and in eucaryotic mitochondria, respectively, and which appear to be virtually identical in form and function). Neupert thus describes methods for post-production modification of the folding of recombinant proteins based on in vitro contact of such denatured recombinant proteins with quantities of heat shock proteins that have been isolated from cells.
Berberian et al. (U.S. Pat. No. 5,348,945) describe methods for enhancement of cell survival under stressful conditions, such methods consisting of in vitro or in vivo application (i.e. addition) of exogenously produced, purified heat shock proteins, such as hsp70, to such stressed cells.
Jacob et al. (U.S. Pat. No. 5,474,892) suggest that certain proteins may be stabilized in aqueous solution via addition of quantities of certain heat shock proteins (such as hsp90). Jacob et al. thus describes post-production methods for modification of the folding or other stabilization of various proteins and other biological materials through in vitro contact of such denatured proteins with quantities of isolated and purified heat shock proteins.
Srivastava (U.S. Pat. No. 5,750,119; U.S. Pat. No. 5,830,464; and U.S. Pat. No. 5,837,251) suggests that tumor proliferation in mammals may be inhibited through inoculation of such mammals with antigenic compounds resulting from association of certain tumor components with various constitutive or exogenous stress proteins. Srivastava therefore describes methods for isolation or formulation of such stress protein/tumor complexes using various tumor specimens, and the subsequent inoculation of mammalian patients with such preparations for the purposes of stimulating anti-tumor response in such patients.
Liu et al. (J. Biol. Chem. 13 (1998) 30704) describe studies of the role of several heat shock proteins, such as hsp40 and hsp70, in enhancement of protein function. In vitro addition of purified exogenously produced heat shock proteins to denatured proteins was reported to lead to enhanced protein function. A co-chaperone role of hsp40 with hsp70 was also noted.
Other pertinent references concerning stress protein function, which are herein incorporated by reference, include:
McGuire et al. (U.S. Pat. No. 5,188,964 and U.S. Pat. No. 5,447,843) describe measurement of the levels of various constitutively produced stress proteins (including the heat shock proteins hsp27, hsp70, and hsp90, and the glucose regulated proteins grp 78 and grp94) present in tumor tissues and use of such measurements as a means for predicting probability of recurrence of such tumors.
Williams et al. (J. Clin. Invest. 92 (1993) 503) describe means for possible protection of cells and tissues from various metabolic stresses, such as ischemia, through transfection with the hsp70 gene. Specifically, constitutively expressed human hsp70 introduced into murine cells enhanced survival of such modified cells upon application of metabolic stress. Addition of such constitutive genes did, however, appear to negatively affect cell proliferation due to the metabolic burden of continual expression, even under unstressed conditions.
Vasconcelos et al. (J. Gen. Virol. 79 (1998)1769) describe means for induction of enhanced expression of constitutive stress protein prior to infection of permissive Vero cell lines with HMV, leading to transient formation of large plaque phenotype variants of HMV. Cells infected 12 hours post stress (where such interval was chosen to allow cells to recover normal physiologic capacity while assuming that induced heat shock protein levels would be maintained over such interval) were found to exhibit slightly enhanced HMV production levels (characterized by up to four-fold increase All in viral titer).
Vasconcelos et al. (J. Gen. Virol. 79 (1998) 2239) describe further means for transient enhancement of stress protein expression through transfection of permissive cell lines (such as human astrocytoma cells) with vectors coding for constitutive hsp72 expression. Clonal cell lines exhibiting uptake of the hsp72 gene were shown to yield increased viral titer upon infection is with HMV. However, such clonal cell lines did not exhibit permanent modification, but rather exhibited a marked tendency to spontaneously regress to the wild-type form. Further, no means for control of expression of the constitutive hsp72 gene is proposed, leading to continuous expression by the modified cell lines. Applicability of the clonal cells produced in this work was limited to HMV attempts to produce other viral products, for example CDV, were unsuccessful. Taken in conjunction with the transient nature of the modification, this suggests limited applicability of this approach.
Yokoyama et. al. (U.S. Pat. No. 5,827,712) describes methods for enhancement of yield in recombinant production of transglutaminase (TG) in E. coli modified for (via transfection) or selected for constitutive overexpression of certain chaperone proteins, including DnaJ and DnaK. Such overexpression is used to stabilize and solubilize functional recombinant TG. Methods for modification include incubation of stress protein vector and TG vector with E. coli. Incubation of a combined stress protein vector/TG vector with E. coli, and incubation of TG vector with E. coli strains exhibiting constitutive overexpression of stress protein. Notably, such methods are likely to yield transient modification wherein such stress protein is continuously produced, negatively affecting cell proliferation due to the metabolic burden of continual stress protein expression.
These examples of prior art clearly establish the role and utility of various stress proteins, such as hsp27, hsp40, hsp60, hsp70 (including hsp72 and hsp73), hsp90, GroEL, GroES, GrpE, grp 78, grp94, DnaJ and DnaK, as aids to enhanced production and stabilization of functional biological materials (such as proteins, viral agents, or other biological agents or products, including enzymes. hormones, growth factors, structural proteins and tumor suppressor agents). However, the methods and concepts taught in these examples, including methods for addition of exogenously produced stress proteins and for induction of transient constitutive production of such proteins, are not sufficiently efficient nor flexible for enhancement of yield in routine production of various biological agents or products, nor for discovery of new biological agents or products. For example, manufacture and purification of useful quantities of stress protein for addition to or modification of product will generally be prohibitively time consuming and expensive. Furthermore, transfection of cells with stress protein vectors coding for constitutively produced stress proteins will, unless incorporating suitable control elements, lead to full-time production of such stress proteins by such cells that will compete with production of desired biological products by such cells. Hence, new, more efficient ways for harnessing and controlling the capabilities and use of such stress proteins are required.
The present invention is directed to new and more efficient methods for harnessing and controlling the capabilities and use of stress proteins in the production and stabilization of functional biological materials, such as proteins, viral agents, or other biological agents or products, including, but not limited to, enzymes, hormones, growth factors, structural proteins, tumor suppressor agents, nucleic acids and nucleic acid probes, vaccines, antigens, antibiotics, lipids, simple and complex carbohydrates, alcohols and other solvents and the products of those methods. Examples of pertinent stress proteins of the present invention include, but are not limited to, hsp27, hsp40, hsp60, hsp70 (including hsp72 and hsp73), hsp90, GroEL, GroES, GrpE, grp 78, grp94, DnaJ and DnaK. In one embodiment of the present invention, the present invention causes the cells to make such hsp""s, as opposed to adding them to the cells.
Specific preferred embodiments of the present invention include, either through the selective induction of constitutive expression of such stress proteins in or through transient or permanent introduction of such stress protein genes into, procaryotic or eucaryotic cells or cell lines, methods for making various non-permissive cell lines permissive, for increasing yield of various biological products, and for discovery and production of various unknown infectious agents.
For example, the use of such embodiments with such cells or cell lines causes dramatic increase in yield of recombinant products in procaryotic or eucaryotic cell lines. This is surprising, since intuitively, one would expect that increasing cellular stress during production would result in decreased productivity. The inventors of the present invention have found, however, that by stressing such cells by certain specific methods or otherwise inducing cellular stress response, thereby leading to controlled expression of stress protein genes, production of functional biological products can be enhanced.
The present invention is directed to such methods and includes the five preferred embodiments specifically illustrated herein. The present invention, however, is not limited to the specifics of these five embodiments but includes modifications and substitutions within the spirit and scope of the invention, as well as other embodiments which will become apparent to those skilled in the art upon reference to this description.
In the first preferred embodiment, transient stress of a eucaryotic cell line is used to enhance viral titer. Permissive eucaryotic cells are transiently stressed for a period sufficient to stimulate production of one or more stress proteins. A desired virus stock is subsequently added following application of this stress so as to infect the stressed eucaryotic cells. Following a period of post-infection incubation, the resulting supernatant, containing the desired virus-induced product, is then harvested.
In the second preferred embodiment, transient genetic modification of a eucaryotic cell line through episomal insertion of a stress protein expression vector, followed by selection of one or more subsets of these modified cell lines that exhibits permanent insertion of the expression vector into host DNA, is used to produce cell lines exhibiting permanent genetic modification. Such cell lines may be used to enhance production of a desired virus or viral product.
In the third preferred embodiment, production of a new permissive eucaryotic cell line is effected through insertion of a stress protein expression vector into a non-permissive eucaryotic cell line. The new permissive cell line is then used to efficiently produce viral agents through inoculation of the cell line with infective or potentially infective material, followed by incubation and harvest of the resultant virus or viral products thereby produced. Such cell lines are preferentially used to facilitate replication of difficult to grow neural agents and those never cultured before. Hence, such lines may be used both as virus hunters and for production or manufacture of useful quantities of agent:
In the fourth preferred embodiment, production of functional recombinant product by genetically engineered procaryotic cell lines is enhanced through insertion of one or more stress protein expression vectors into such cell lines. It is preferred that recombinant cells be selected for use. However, clonal cells may also be selected. It is further preferred that such inserted stress protein expression vectors include one or more inducible promoter. Alternatively, a constitutive promoter can be used. Expression of such stress protein expression vectors, either by induction or by constitutive expression, in such cell lines results in production of the one or more coded stress protein, wherein such expressed stress protein thereby serves to assist in enhancement of yield of functional recombinant product.
In the fifth preferred embodiment, production of functional recombinant product using genetically engineered eucaryotic cell lines is enhanced through insertion of one or more stress protein expression vectors into such cell lines. Such insertion may be effected prior to or after genetic modification of the line for production of the desired recombinant product. It is preferred that such stress protein expression vectors include one or more inducible promoter. Alternatively, a constitutive promoter can be used.